Elsevier

Cellular Signalling

Volume 20, Issue 10, October 2008, Pages 1855-1864
Cellular Signalling

NMDAR-nNOS generated zinc recruits PKCγ to the HINT1–RGS17 complex bound to the C terminus of Mu-opioid receptors

https://doi.org/10.1016/j.cellsig.2008.06.015Get rights and content

Abstract

In neurons, the C terminus of the Mu-opioid receptor (MOR) binds to the protein kinase C-interacting protein/histidine triad nucleotide binding protein 1 (PKCI/HINT1) which in turn binds the regulator of G-protein signalling RGSZ1/Z2 (RGSZ) protein. In this study, we found that intracerebroventricular (icv) administration of morphine recruits PKC isoforms, mostly PKCγ, to the MOR via the HINT1/RGSZ complex. There, diacylglycerol (DAG) activates this PKCγ to phosphorylate the MOR and thus, its signal strength was reduced. When PKCI/HINT1 expression is depressed, morphine produces stronger analgesic effects and neither the PKCγ–MOR complex nor serine phosphorylation of this receptor is detected. This MOR–PKC association involves the cysteine rich domains (CRDs) in the regulatory C1 region of PKC, as well as requiring free zinc ions, HINT1 and RGSZ proteins. Increasing the availability of this metal ion recruits inactive PKCγ to the MOR, while phorbol esters prevent this binding and even disrupt it. The nitric oxide donor (S)-Nitroso-N-acetylpenicillamine (SNAP) foments the association of PKCγ with the MORs, effect that was prevented by the heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), suggesting a role for endogenous zinc and neural nitric oxide synthase. The N-methyl-D-aspartate receptor (NMDAR) antagonist, MK801, also prevented PKCγ recruitment to MORs and serine phosphorylation of the receptors following icv morphine. These results indicate that the NMDAR/nNOS cascade, activated via MORs, provide the free zinc ions required for inactive PKCγ to bind to HINT1/RGSZ complex at the C terminus of the receptor.

Introduction

The MOR associates with a series of proteins that help propagate the information carried by its agonists and that regulate the strength of their signals [1]. Besides the Gi/o proteins, this opioid receptor also regulates the Gα subunit of Gz proteins [2], [3]. The Gαz is virtually restricted to the nervous system [see references in 4] and its GTPase activity is extremely inefficient, about 200-fold slower than that of the Gαi/o subunits. Thus, it requires the assistance of specific GTPase activating proteins to augment its rate of hydrolysis [5], [6]. In the nervous tissue, the MOR is associated to a signalling module that helps control the activity of agonist-activated Gαz subunits. The C terminus of this opioid receptor binds the protein kinase C-interacting protein (PKCI), which also associates with specific GTPase activating proteins (GAPs) of receptor-activated GαzGTP subunits, the RGS17(Z2) and RGS20(Z1) proteins [7], [8]. PKCI is a 126 amino acid protein that displays zinc-specific binding [9], and can act as a nucleotidyl hydrolase or phosphotransferase [10]. PKCI is also known as the histidine triad nucleotide binding protein 1 (HINT1), a member of the histidine triad (HIT) protein family characterized by the sequence HisXHisXHis (where X is a hydrophobic amino acid). PKCI/HINT1 exists as an homodimer that does not require zinc ions to associate [11], and it interacts with a series of proteins [12], protein microphthalmia transcription factor [13], cyclin-dependent kinase 7 [14], pontin and reptin [15].

At the MOR, the PKCI/HINT1 connects the C terminus of this receptor with the N-terminal cysteine rich domain (CRD) of RGSZ1 and RGSZ2 proteins, their CRDs binding to the conserved triad of histidines at the C terminus of the PKCI [7]. In the absence of the appropriate GAP, the regulatory activity of GαzGTP on effectors increases the potency of MOR agonists such as morphine but afterwards, it brings about a profound desensitization of the receptors [16], [17]. A similar pattern of morphine potentiation followed by severe tolerance has been observed for PKCI knockout mice [8]. Thus, the association of HINT1/RGSZ with the MOR appears to control (reduce) the activity of morphine and to prevent profound desensitization of the MORs.

The PKCI/HINT1 was initially described as a potent inhibitor of PKC [18], a serine/threonine kinase implicated in the development of tolerance to MOR agonists [reviewed in 19]. The C1 regulatory domain of the PKCs is a CRD that is found in all PKC isozymes. In conventional PKCα/β/γ and novel PKCδ/ε/θ/η(L) this domain is organized as a tandem C1A and C1B repeat that coordinates the binding of four zinc atoms, as well as binding phorbol esters or diacylglycerol [20]. The binding of zinc to the C1 CRDs greatly facilitates the subsequent binding of phorbol esters and the activation of the PKC, as well as its translocation to the plasma membrane [21], [22], [23], [24]. For some of these conventional PKCs the CRD2 has been shown to associate with other proteins [25]. These observations, and the finding that phorbol-12-myristate-13-acetate (PMA)-induced phosphorylation of MOR was reduced by the presence of PKCI/HINT1 [8], suggest that this protein also reduces the inactivating action of PKC on the MOR.

Thus, to gain further knowledge on the specific mechanisms that regulate the activity of the MOR in the nervous system, we have investigated whether MOR-associated HINT1/RGSZ recruits PKC to regulate the signalling activity of morphine. We also assessed the role of zinc in this process, and whether the glutamate NMDA receptor (NMDAR)-neural nitric oxide synthase (nNOS) cascade might be the physiological source of the cytosolic free zinc ions required.

Section snippets

Preparation and solubilization of synaptoneurosome-enriched fractions of mouse PAG

Experimental tissue was obtained from male albino CD1 mice (Charles River, Barcelona, Spain) weighing 22–27 g. The mice were sacrificed by cervical dislocation and the periaqueductal grey matter (PAG) was removed. About 1 mm of tissue around the aqueduct was collected from 2 mm thick coronal sections (MP-600 micropunch, Activational Systems Inc.) and the structures from six to eight mice were used in a typical immunoprecipitation assay (e.g., at one post-morphine interval). The assays were

Influence of PKCI/HINT1 knockdown on the supraspinal analgesia produced by icv-injection of morphine to mice

The murine PKCI/HINT1 protein contains 126 amino acids and in PAG synaptosomes, it is detected as a band of about 14 kDa compatible with the monomer (Fig. 1), and also as 28 kDa protein that could correspond to the dimer [11] or to a form carrying attached the small ubiquitin-like modifier (SUMO). Antisense oligodeoxynucleotides (ODNs) have probed effective in reducing the expression of G-protein-coupled receptors, including opioid receptors, and associated signalling proteins in vivo. The ODNs

Discussion

The MORs in the nervous system regulate the tissue-specific signalling protein Gz, and couple to its selective regulators the RGSZ1 and RGSZ2 through the PKCI/HINT1 protein. This adaptor protein connects the cytosolic C terminus of the MOR with the cysteine rich domain present in these RGSZ proteins [7], thereby participating in the control of activated GαzGTP subunits which are able to promote receptor desensitization [16], [17]. In the present study, we found that the HINT1/RGSZ complex

Conclusions

This study identifies the molecular process by which PKC, and in particular PKCγ, diminishes the response of morphine-activated MORs, facilitating their desensitization to further doses of the opioid. This is achieved by a two step process, in which the activated MORs first promote an increase in cytosolic free zinc. This metal ion translocates and stabilizes inactive PKCγ to the cytosolic tail of the receptor by binding to the HINT1–RGSZ complex, bringing the kinase close to its target, the

Acknowledgements

The research was supported by the Spanish Ministerio de Educación y Ciencia (SAF2005-01772 and SAF2006-03193). Elena de la Torre-Madrid is a predoctoral fellow from Ministerio de Educación y Ciencia (FPI). We would like to thanks Beatriz Fraile for her excellent technical support.

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